The interaction of the erythroid transcription factor, GATA-1 with DNA is a major focus of our research. Vertebrate GATA factors have two zinc fingers that comprise the DNA binding domain. The C-terminal finger is the main DNA binding finger and the three dimensional structure of this finger of GATA-1 bound to DNA has been solved by NMR. The structure reveals a finger and helix that bind to the major groove of DNA and an adjacent basic arm that binds in the minor groove. However, the GATA-1 N-terminal finger also plays an important role in DNA binding and in the function of the protein. Mutations in the N-finger that interfere with its ability to bind to DNA are associated with thrombocytopenia and thalassemia in humans and anemia in transgenic mice. The two zinc fingers of GATA-1 interact with each other in ways that can lead either to enhancement or to inhibition of DNA binding, depending on the sequence of the binding site. We have shown that GATA-1 adopts different conformations that are binding site specific, and these variations can be detected by altered migration in electrophoretic mobility shift assays or by differential resistance to proteases. These variations are not due to DNA bending since we have established that GATA-1 bends DNA slightly in a binding site independent manner. We have also demonstrated that GATA-1 is unable to stimulate transcription when bound to some DNA sites, suggesting allosteric regulation. Several crucial cofactors that interact with the zinc fingers of GATA-1 have been identified, and their ability to bind to GATA-1 may be influenced by the conformation that GATA-1 adopts in response to DNA. Consequently, we are attempting to solve the structure of the linked GATA-1 zinc fingers on a number of DNA binding sites by Xray crystallography. With one binding site, we have co-crystals that diffract to 3.1 angstroms and we are trying to determine the phase of the crystals with iodinated and brominated DNAs and with MADS experiments with the native crystals. Meanwhile we have taken a biochemical approach to show that the N-terminal finger interacts with DNA in a manner similar to the C-finger. Three amino acids in the C-finger helix make base specific contacts with DNA, and we have shown that two analogously positioned N-finger amino acids are required for N-finger binding to DNA. This strongly suggests that he mode of DNA recognition is similar for both fingers. In addition, the binding specificity of the N- and C-terminal fingers may be somewhat different. Binding site selection experiments using the GATA-1 C-finger, the GATA-1 N-finger fused to the basic arm of the C-finger, and the GATA-2 N-finger, show that the N-finger of GATA-2 prefers sites containing GATC while both of the GATA-1 fingers tested prefer GATA containing sites. Recently we have found that the N-finger of GATA-1 also binds to DNA, but with such low affinity that a selection experiment could not be performed. However, conventional binding studies have shown that this finger prefers the GATC site and that,as noted above, the C-terminal basic arm can change its specificity to GATA. Fusing this same basic arm to the GATA-2 N-finger also changes its specificity from GATC to GATA, suggesting that the GATA-1 basic arm controls the specificity at the last base of the core binding site. We are identifying the amino acids involved in these preferences. Because some biologically important GATA binding sites contain the GATC sequence, the mode of DNA recognition at these sites is significant. The N-finger of the GATA proteins may be particularly important at these sites. DNA recognition by the N-finger of GATA-3 is also important for the regulation of some genes. Three GATA recognition sequences in the IL13 gene promoter form a high affinity binding site for two molecules of GATA-3 and two of the sites have GATG as their core sequence. All three sites are necessary for full activity of this promoter at limiting GATA-3 concentrations and the N-finger is involved in binding to these sites. The IL 5 gene also contains some palindromic GATA binding sites that may be important for gene expression and require the N-finger of GATA-3.